Responses to saltwater exposure vary across species, populations and life stages in anuran amphibians

Abstract To predict the impacts of environmental change on species, we must first understand the factors that limit the present-day ranges of species. Most anuran amphibians cannot survive at elevated salinities, which may drive their distribution in coastal locations. Previous research showed that coastal Hyla cinerea are locally adapted to brackish habitats in North Carolina, USA. Although Hyla squirella and Hyla chrysoscelis both inhabit coastal wetlands nearby, they have not been observed in saline habitats. We take advantage of naturally occurring microgeographic variation in coastal wetland occupancy exhibited by these congeneric tree frog species to explore how salt exposure affects oviposition site choice, hatching success, early tadpole survival, plasma osmolality and tadpole body condition across coastal and inland locations. We observed higher survival among coastal H. cinerea tadpoles than inland H. cinerea, which corroborates previous findings. But contrary to expectations, coastal H. cinerea had lower survival than H. squirella and H. chrysoscelis, indicating that all three species may be able to persist in saline wetlands. We also observed differences in tadpole plasma osmolality across species, locations and salinities, but these differences were not associated with survival rates in salt water. Instead, coastal occupancy may be affected by stage-specific processes like higher probability of total clutch loss as shown by inland H. chrysoscelis or maladaptive egg deposition patterns as shown by inland H. squirella. Although we expected salt water to be the primary filter driving species distributions along a coastal salinity gradient, it is likely that the factors dictating anuran ranges along the coast involve stage-, species- and location-specific processes that are mediated by ecological processes and life history traits.


Introduction
Anthropogenic environmental change is progressively altering habitats around the globe (Pörtner et al., 2022). To accurately predict species responses to impending habitat changes, we need to understand how organisms have adapted to historic and contemporary environmental conditions and how these biological responses affect habitat use and species ranges (Urban et al., 2016). However, the factors that determine range limits are ambiguous for many species, which hinders our ability to forecast whether future conditions will induce habitat shifts and range expansions or contractions (Holt, 2003;Parmesan et al., 2005). Species ranges reflect complex interactions between biotic, abiotic, spatial and historical factors (Gaston, 2009;Roy et al., 2009), but the physical environment often serves as a primary filter determining a species presence because populations cannot persist in an environment that reduces the intrinsic rate of growth (λ) below one (Holt, 2003). Therefore, understanding how changes in key abiotic factors in the environment affect organismal performance will improve understanding of current species ranges, and how species ranges may change in the future in response to progressive environmental change (Gaston, 2009).
One important abiotic filter is the concentration of salts in the environment, which is illustrated by the abrupt demarcation of range boundaries between most marine and freshwater species. Although salinization of freshwater ecosystems can occur naturally from the weathering of rock and intrusion by saline groundwater, anthropogenic activities are rapidly increasing salt concentrations (Hintz et al., 2022). Overextraction from coastal freshwater aquifers, mining and gas extraction, road de-icing salts, surface water diversions, changes in rainfall patterns and sea level rise are all contributing to increases in salinity of freshwater systems (Herbert et al., 2015). Furthermore, salts can remain and accumulate in ecosystems for years (Kelly et al., 2008;Herbert et al., 2015;Kaushal et al., 2018), and the economic costs of wetland remediation can be prohibitively expensive (Hintz et al., 2022). As a result, an increasing number of species are likely to be affected by salt-intruded wetlands (Hintz and Relyea, 2019). Therefore, there is a growing need to understand the impacts of salinization on freshwater biodiversity.
Changes in salinity can be physiologically stressful to freshwater organisms that even modest increases in salinity can generate consequences that cascade throughout entire ecosystems (Jones et al., 2017;Verhille et al., 2020;Arnott et al., 2023). Anuran amphibians (frogs and toads) are osmotically sensitive due to highly permeable ectodermal membranes, although there is more variation in salinity tolerance across amphibia than is commonly appreciated (Hopkins and Brodie, 2015;Albecker and McCoy, 2017). More than 140 anuran species have been observed in saline wetlands (Hopkins and Brodie, 2015), although few species have been observed in full-strength seawater (e.g. Bufo viridis, Fejervarya cancrivora; (Gordon et al., 1961;Gordon, 1962). Moreover, studies have indicated that some anuran populations have altered salt tolerance via local adaptation (Gomez-Mestre and Tejado, 2003;Hall et al., 2017; or maladaptation (Brady, 2013;Forgione and Brady, 2022). Therefore, changes in wetland salinity may cause shifts in habitat usage and range limits for some anuran species.
Anurans have a complex life cycle, in which they develop sequentially into distinct morphological forms that are accompanied by changes in ecological niche (Wilbur, 1980;Werner, 1988). Therefore, the effects of salt water on amphibian survival, physiology, development and behaviour depends on which life history stage is exposed (Karraker, 2007;Karraker et al., 2008;Albecker and McCoy, 2017). In most North American species, the egg and larval stages (i.e. tadpoles) are fully aquatic, which makes those life stages more likely to be affected by wetland salinity than terrestrial adults. Sublethal exposure to physiological stressors during larval life stages can slow growth and development because the physiological costs associated with maintaining homeostasis usurps resources for growth and disrupts developmental pathways (Gomez-Mestre et al., 2010;Burraco and Gomez-Mestre, 2016;Hall et al., 2017;Szeligowski et al., 2022). However, many aquatic organisms with complex life histories have evolved different strategies to balance the tradeoff between larval growth and development and mortality due to historic selection pressures like pond desiccation and predation (Albecker et al. 2023;Wilbur and Collins, 1973;Werner, 1986). For instance, some species avoid laying eggs in permanent water bodies because they are more likely to contain fish predators that can cause population declines (Rieger et al., 2004). Anuran clades that evolved to primarily inhabit ephemeral ponds (ephemeral species) accelerate developmental rates at the expense of growth (Richter-Boix et al., 2011;Pujol-Buxó et al., 2016). Ephemeral species are hypothesized to sustain growth rates near physiological capacity to increase the likelihood of reaching minimum size thresholds before pond drying, so it is possible that operating at physiological extremes may increase vulnerability to environmental stressors relative to species that develop in more permanent water bodies (Richter-Boix et al., 2011). Therefore, differences in life history strategy that evolved in response to historical selection pressures like predation and hydroperiod could also affect vulnerability to contemporary environmental stressors like salinity.
In this study, we investigated how saltwater exposure affected different populations and life stages of three congeneric tree frog (family Hylidae) species (Hyla cinerea, Hyla squirella and Hyla chrysoscelis) that have similar life histories but use water bodies with different salinities and hydroperiods (Albecker and McCoy, 2017). Although all three species are common, abundant and sometimes occur sympatrically in inland ponds, there is variation in the microgeographic distributions of these species in North Carolina (NC, USA) (Albecker and McCoy, 2017 (Albecker and McCoy, 2017). Importantly, H. cinerea breed in both permanent and semi-permanent (i.e. long hydroperiod) ponds and would thus qualify as a 'permanent pond breeding' species, whereas H. squirella exploit temporary ponds for reproduction and typically do not breed in long hydroperiod wetlands that can contain fish. Finally, H. chrysoscelis breeds in both permanent and temporary ponds and co-occurs with both H. cinerea and H. squirella at inland freshwater sites. However, H. chrysoscelis have not been observed in coastal, brackish wetlands and are far less abundant than H. cinerea and H. squirella in coastal locations in North Carolina (Gibbons and Coker, 1978).
Given the dissimilarity in the coastal distribution of these phylogenetically closely related species, we investigated how these species responded to saltwater exposure and whether differences in natural exposure to salt water between populations sourced from coastal and inland locations influenced their responses. We tested how exposure to salt water affected breeding behaviour (e.g. oviposition site choice), hatching rates and early larval survival, body condition (size) and plasma osmolality. We expected to observe greater vulnerability to salt stress among H. chrysoscelis, H. squirella and inland H. cinerea and the least vulnerability in coastal H. cinerea. If a life history tied to ephemeral ponds contributed to physiological vulnerability, we expected H. squirella to demonstrate the lowest salt tolerance. We anticipated that growth and plasma osmolality data would reflect differences in salt sensitivity, with higher plasma osmolality and increased growth shown by species and populations with higher survival and hatching rates in salt water. Finally, we only expected to observe population-level differences in H. cinerea based on results from previous work demonstrating salt adaptation in coastal populations of H. cinerea McCoy, 2017, 2019).

Methods
This study was conducted in eastern North Carolina, USA, between May 4, 2016, and July 24, 2016. Eastern North Carolina is an important location for investigating interspecific and intra-specific variation in responses to saltwater exposure because this region is affected by overwash and storm surges from coastal weather systems and is projected to experience >1 m of additional sea level rise over the next century (Anthony et al., 2009;Craft et al., 2009). Climate change-driven increases in coastal flooding and saltwater intrusion into coastal wetlands are already having strong impacts on this region (Cowart et al., 2010;Kopp et al., 2015;Covi et al., 2021).
To characterize how responses to salt water differ among species and locations, we compared the responses of anuran species (H. cinerea, H. squirella and H. chrysoscelis) collected from multiple populations from coastal and inland locations. We do not include any coastal H. chrysoscelis populations because no coastal breeding aggregations were identified during the study. Coastal populations and inland populations used in this study were geographically separated from one another by ∼200 km. All protocols for these experiments were approved by East Carolina University's Animal Care and Use committee (D328 and D314), and animals were collected under North Carolina Wildlife Collection License (no. 16-SC00840).
Experimental Methods: We tested oviposition site choice by collecting amplexed pairs from both coastal or inland populations and followed the experimental protocols outlined in Albecker and McCoy (2017). On capture, each pair was placed into an 18-liter clear plastic bin that contained six square pint cups (10.8 cm width × 6.4 cm height) filled with 400 ml of water. Three randomly selected cups contained treated tap water (0.5 ppt), and the other three cups contained salt water prepared by mixing treated tap water with InstantOcean Sea Salt ® (Blacksburg, VA). Therefore, each bin presented breeding pairs with a binary choice between laying eggs in fresh water or salt water, with different bins containing a different concentration of salt water (e.g. 4, 6, 8 or 12 ppt). Each replicate contained all four salt concentration treatments (1 replicate = 4 bins with one breeding pair per bin), with each replicate arranged in a spatial block at the site of collection. Bins were left undisturbed overnight at the breeding site to allow oviposition. The following morning, adult frogs were released, lids fastened to each cup to prevent mixing of water or eggs, and bins were transported to the laboratory. Eggs within each cup were photographed, the salinity remeasured and the eggs were monitored for hatching. Hylid eggs are typically laid in floating masses of single or double layers, which allows for eggs to be easily counted from photographs using ImageJ software (Schneider et al., 2012). Eggs hatched between 48 and 96 h after oviposition. At this point, the salinity of the pint cup was measured again, and hatchlings were counted and recorded.
To determine the effects of salinity on early larval survival, we used the individuals that hatched from freshwater cups. We only used hatchlings from fresh water to avoid confounding embryonic exposure to salt water with any downstream results. Hatchlings were held in the original cups in the laboratory (26.67 • C) and allowed to develop until reaching Gosner stage 25, which took ∼48-72 h after hatching (Gosner, 1960). At this point, we aggregated all tadpoles from the freshwater cups from each clutch (all individuals from a single bin represented one clutch) and then haphazardly sorted individuals into five groups of fifty tadpoles, which were then randomly assigned to a salinity treatment. Each clutch comprised a single replicate block to account for potential genetic effects. We placed these groups into 350-ml glass dishes containing 300 ml of treated tap water within a laboratory with a 12-h light/dark cycle. After acclimatizing for 24 h, salinities were incrementally raised to one of four target salinities (e.g. 4, 6, 8 and 12 ppt) through 6 d to reduce osmotic shock and reflect natural variation in salinity in coastal wetlands in which  (Hsu et al., 2012;Albecker et al., 2018;. Freshwater treatments were maintained at 0.5 ppt throughout. Tadpole mortality in each cup was assessed daily and recorded, and all deceased individuals were removed. To perform daily water changes, tadpoles were carefully collected into a small fish net and carefully redeposited into clean water. Tadpoles were fed 10 mg of Spirulina fish food flakes (Ocean Star International, Coral Springs, FL) each day after water changes.
At the conclusion of the 6-d salinity exposure period, final survival was recorded. At this time, 20 surviving individuals from each cup were haphazardly selected using a transfer pipette and euthanized via 2% MS-222 immersion (pH adjusted to 7.0). Ten of those individuals were staged, weighed and total length measured (snout to tip of tail). All tadpoles across species, locations and treatments were between Gosner stages 26 and 28 (Gosner, 1960). The remaining ten tadpoles were blotted dry using paper towels, each placed into 2-ml tubes, homogenized using mechanical mortar and pestle and centrifuged for 2 min (Lai et al., 2019). Supernatant was pipetted into a test tube and plasma osmolality was measured using a Fiske 210 micro-osmometer from Advanced Instruments.
Statistical Methods: Analyses were conducted in the R statistical programming environment version 4.0.5. We analysed the proportion of egg clutches that were laid in fresh water (i.e. oviposition site choice), the probability of hatching, the proportion of eggs that hatched, tadpole survival on the final day of acclimations, tadpole size and whole-body plasma osmolality. For tadpole size, we estimated body condition, which is mass corrected for differences in body length. We calculated an index of condition by dividing mass by total length raised to the power of the slope of the relationship between log transformed mass and length (Peig and Green, 2009). Because the slopes were similar across all species and locations, we estimated and applied a single slope to data from all species-locations using a simple linear model in package "lme4" (Bates et al., 2014).
For each analysis, we used a model selection approach by first fitting generalized linear mixed effects models (GLMMs) in package "lme4" (Bates et al., 2014) and then evaluating levels of support for different models ranging from most complex (i.e. full interaction between fixed effects) to simpler (i.e. additive) and random effects-only models using likelihood ratio tests (Burnham and Anderson, 2003). We considered salinity and species-location pairs (e.g. inland H. cinerea, coastal H. cinerea, etc.) as fixed effects in each model. Hatching data were overdispersed so we used a two-step hurdle approach for these data. First, we analysed the probability of hatching using a binary response [0 if no eggs hatched (total clutch failure); 1 if any egg hatched]. We then excluded the cups in which no eggs hatched and ran a subsequent model to understand how the proportion of eggs that hatched varied according to species-location pair and salinity. We investigated larval survival using two complementary analyses: First we tested for overall differences in the proportion of surviving tadpoles according to salinity and species-location by filtering the data to just the final day. Then, we tested whether there were differences in survival through time in the highest salinity treatment (12 ppt) according to the day and species-location (both as fixed effects).
We included random effects in each model. For the proportion of eggs laid in fresh water and hatching hurdle models, we treated each experimental bin as a random effect, whereas for analyses on tadpole survival, we treated the individual cup in which tadpoles were housed as a random effect. Finally, for plasma osmolality and body condition, we treated replicate as a random effect. We assumed binomial error distributions with logit link for the proportion of eggs laid in fresh water (including total number of eggs as weight), hatching probability, hatching proportion (with total eggs as weight) and tadpole survival analyses (with total number of tadpoles as weight). We assumed lognormal error for body condition and a Poisson error distribution for plasma osmolality (Bolker, 2008). For all mixed effects models, we used the bobyqa optimizer.

Results
The proportion of eggs laid into fresh water was best described by a model that included the interaction between salinity and species-location pair (χ 2 4 = 20.54, P = 0.0004). In the lowest salinity (4 ppt), pairs from all species and locations deposited ∼68% of their eggs into fresh water (Fig. 1). As salinities increased, coastal H. squirella, coastal and inland H. cinerea and inland H. chryscoscelis increased the proportion of eggs laid into fresh water to ∼83% in the highest salinity (12 ppt). However, inland H. squirella showed the opposite trend and reduced the proportion of egg clutches deposited into fresh water as salinity increased (∼24% in fresh water in 12-ppt treatment; Fig. 1).
There was a significant interaction between salinity and species-location explaining the probability that any eggs hatched (χ 2 4 = 10.07, P = 0.039; Fig. 2A). Hyla chrysoscelis had the lowest probability of hatching relative to other species (Fig. 2B), with a 50% probability of total clutch loss in ∼3.3 ppt of salt water. All other species showed a 50% probability of total clutch loss between 5.3 and 6 ppt of salt water. In the same salinity (5.4 ppt), H. chrysoscelis had just 10% probability of hatching. After excluding cups in which no hatching occurred, we also found an interaction between salinity and species-location on the proportion of eggs that hatched (χ 2 4 = 360.65, P < 0.001; Fig. 2C). In general, the proportion of eggs that hatched decreased as salinity increased. Coastal H. squirella had the highest hatching rates in fresh water (∼80%), but also had the most rapid decline in hatching proportion as salinities increased, with just 25% hatching in the 6-ppt treatments. Fifty percent of H. cinerea embryos from coastal and inland locations hatched in 6 ppt of water, whereas 60% hatched in 6 ppt for inland H. squirella. Interestingly, H. chrysoscelis showed no decline in hatching proportion, with ∼70% hatching in all salinities.
Tadpole survival on the final day of salinity acclimations was best described by an interaction between salinity and species-location (χ 2 4 = 461.56, P < 0.001; Fig 3A). Inland H. squirella showed the highest survival in the highest salinity (12 ppt), with ∼66% of individuals surviving, whereas coastal H. squirella netted 44% survival in the 12ppt treatment. Coastal H. cinerea had higher survival than inland H. cinerea across salinities, which was most apparent in the 8-ppt treatment (88% survival vs 66% survival, respectively). Notably, in the highest salinity, H. cinerea from coastal and inland locations had lower survival than H. squirella from both locations (inland H. cinerea, ∼20% survived; coastal H. cinerea, ∼28% survived). Hyla chrysoscelis survival remained ∼90% through 8 ppt, with survival rates in the highest salinity comparable with coastal H. cinerea. We observed similar patterns in survival through time in the 12-ppt treatment, in which an interaction between day and species-location best described the proportion of surviving individuals (χ 2 4 = 206.47, P < 0.001; Fig. 3B). Across all species-location groups, survival remained high (>85%) until day four. On day four (at which point salinities reached 8 ppt), inland H. cinerea survival dropped to 64%, coastal H. squirella fell to 80%, whereas the other species-locations remained at ∼90% survival. Inland H. cinerea continued to show a steep decline to just 20% survival on the final day (day six), whereas other species-locations demonstrated a shallower dip in survival on days five and six (Fig. 3).
There was an interaction between salinity and specieslocation on body condition (χ 2 4 = 12.28, P = 0.015; Fig. 4A). Hyla chrysoscelis had the lowest body condition and their condition remained largely unchanged across salinities. Hyla cinerea had slight increases in condition as salinities increased, with coastal populations having slightly larger condition than inland frogs. Hyla squirella had the largest condition indices of the three species in fresh water, but their condition index decreased as salinities increased.

Discussion
Anuran amphibians are not commonly observed in saline or brackish environments, but variations in salt tolerance exist across species and across populations. In a previous field study, we observed that H. cinerea was abundant along the North Carolina outer banks coast and regularly occupied brackish habitats (Albecker and McCoy, 2017). Hyla squirella is also abundant in coastal locations but is observed only in rain-filled freshwater ephemeral wetlands. Hyla chrysoscelis was far less abundant in coastal locations and was only observed in upland freshwater wetlands within coastal hardwood forests. Furthermore, there are no published accounts of H. squirella or H. chrysoscelis occupying saline wetlands (Hopkins and Brodie, 2015). We predicted that saltwater tolerance would vary among these species, with H. cinerea being the most salt-tolerant, and that differences in salt tolerance would be a primary factor driving the observed differences in habitat occupancy along the coast. Contrary to expectations, coastal H. cinerea did not have greater salt tolerance than the other species and locations. In fact, if responses shown by coastal H. cinerea are considered the reference point for salt tolerance, the results from this study indicate that all three species across inland and coastal locations may be able to survive in saline wetlands. Thus, the mechanisms underlying the microgeographic variation in coastal locations for H. squirella and H. chrysoscelis are unlikely to be mediated by wetland salinity alone. Adaptive responses to other historical selection pressures may limit occupancy of saltwater wetlands. For instance, H. squirella and H. chrysocelis typically breed in ephemeral or temporary water bodies that form in shallow basins after rain because periodic drying prevents colonization by fish predators (Resetarits and Wilbur, 1989;Rieger et al., 2004;Binckley and Resetarits, 2008). Brackish coastal wetlands tend to be permanent or tidal and almost always contain fish; thus, H. squirella and H. chrysocelis may not inhabit these systems because they lack favorable ecological cues and geophysical properties, rather than simply because they contain salt. Multiple studies have shown that coastal populations of H. cinerea are locally adapted to tolerate and use brackish habitats (Schriever, 2007;McCoy, 2017, 2019), and this species is abundant in coastal habitats across their range in the southeastern USA (Hardy, 1953;Neill, 1958;Tuberville et al., 2005;Gunzburger, 2006;Schriever, 2007). Therefore, we expected coastal H. cinerea to exhibit higher embryonic hatching success and higher larval survival in salt water relative to all other groups including inland H. cinerea, H. chrysoscelis and H. squirella. However, coastal H. cinerea did not show a higher probability of hatching or an increased proportion of egg clutch hatching (Fig. 2). Furthermore, the survival of coastal H. cinerea tadpoles in the highest salinity treatment (12 ppt) was markedly lower than that of coastal and inland H. squirella tadpoles in 12-ppt treatments (∼25% survival vs ∼65% survival) and roughly equaled the survival of inland H. chrysoscelis tadpoles. Collectively, these results indicate that coastal H. cinerea does not have any clear advantage in saltwater tolerance in the early life stages over these congeners.
Previous work by the authors has explored the pathways of local adaptation in coastal populations of H. cinerea Albecker et al., 2021), and we not only expected H. cinerea to display exceptional salt tolerance, but also expected parallel differences between coastal and inland populations of H. cinerea in this study. We observed higher plasma osmolality and tadpole survival in coastal populations than in inland populations, which is consistent with previous studies McCoy, 2017, 2019). However, differences in hatching probability, proportion of egg clutches that hatched, proportion of eggs laid in fresh water and body condition of coastal and inland H. cinerea populations were less pronounced in this study. This suggests that other unaccounted-for environmental factors can mitigate responses to salinity, which might make it a more variable force than previously assumed. Nonetheless, these data confirm increased salt tolerance among coastal H. cinerea populations during the larval stage, which may offer some protection against future variations in salinity.
We expected that the life history of H. squirella might contribute to the differences in coastal habitat use. Specifically, H. squirella is a burst-breeding, fast-developing species that commonly forms breeding aggregations in temporary, fishless ponds and wetland depressions after heavy rains. The dependence on ephemeral rain-filled wetlands has led to an evolved strategy to maximize developmental rates, possibly to the brink of physiological capacity (Richter-Boix et al., 2011). As a result, maximizing development may impose a costly energy trade-off that increases vulnerability to abiotic stress. In support of this, coastal H. squirella showed the most rapid and pronounced decline in the proportion of egg clutches that hatched as salinity increased (Fig. 2C). However, this heightened vulnerability was shortlived because the subsequent experiment on early tadpole survival showed an extraordinary tolerance for salt water, with much higher survival in 12-ppt water than in saltadapted, coastal H. cinerea (Fig. 3). Our study suggests that if there is a relationship between rapid development and sensitivity to abiotic stressors, fast-developing species may be evolutionarily primed to tolerate highly variable or stressful environments. In addition, the observed differences in saltwater tolerance shown by coastal and inland H. squirella may have an epigenetic basis. For instance, it is possible that inherited epigenetic patterns in coastal locations in response to salt exposure in previous generations impose significant energetic burdens on developing coastal larvae (Mueller et al., 2016;Jeremias et al., 2018). However, the role of epigenetics in the maintenance of osmoregulatory homeostasis throughout development is poorly understood in amphibians and requires more research. Finally, the avoidance of saltwater habitats by coastal H. squirella may also be driven by evolved predator avoidance strategies and the geophysical characteristics of the habitats. We assumed that differences in habitat choice would be driven by the physiological limits of the species, but there are also important ecological differences among these types of habitats. For instance, preferential breeding in ephemeral ponds allows H. squirella to avoid fish predators that are present in the more permanent hydroperiod marshes occupied by H. cinerea, a behavioural oviposition choice that has been previously observed in this species Resetarits, 2002, 2008).
Similarly, we anticipated that H. chrysoscelis would show reduced larval survival in salt water, but instead, this species showed larval saltwater tolerance that was on par with salt-adapted coastal H. cinerea (Fig. 3). However, H. chrysoscelis seemed to be more sensitive to salt water during the embryonic stage, with a 50% probability of total clutch failure in 3 ppt, whereas the other species and locations had a 50% probability of total clutch failure in 6-ppt water ( Fig. 2A). The higher vulnerability at the egg stage may contribute to their exclusion from brackish coastal habitats where total clutch failure may be more likely. However, when total clutch failure did not occur for H. chryoscelis, most of the clutch (∼75%) successfully hatched, even at higher salinities (Fig. 2C). Nonetheless, population-level dynamics that result in mortality at the egg stage are likely mediated by density-dependent regulation occurring during larval life stages, which can hinder our ability to infer populationlevel consequences based on egg mortality alone (Vonesh and De la Cruz, 2002). These phenomena may be further mediated by interactions between density dependence and salt sensitivity. For instance, high densities (such as those used in our larval survival experiment) could modify responses to salinity stress differently across species (Albecker et al., 2020). Finally, like H. squirella, H. chrysoscelis also breeds in ephemeral wetlands and avoids habitats containing fish, so evolved adaptations to avoid fish-containing habitats may also contribute to their absence from brackish systems (Resetarits and Wilbur, 1989 Earlier work suggested that increases in blood plasma solute concentrations allows some salt-tolerant amphibian species to remain hypertonic to the external environment and thus tolerate high salinities (Gordon et al., 1961;Gordon and Tucker, 1965;Balinsky, 1981). For example, the Asian crab-eating frog, Fejervarya cancrivora, tolerates nearly fullstrength seawater by accumulating high plasma solute concentrations of urea, sodium and chloride that preserves the hyperosmotic relationship between the frog and its external environment (Gordon et al., 1961;Wu and Kam, 2009;Wu et al., 2014). Although many anuran species do not show the same ability to accumulate and tolerate plasma solute concentrations, modest increases in plasma solute concentrations are hypothesized to reduce the osmotic gradient between individuals and the environment, which may buffer against fatal amounts of water and ion flux (Wells, 2007). Thus, we may expect higher survival rates in species that have high plasma solute concentrations because their bodies remain hypertonic to the environment across a larger range of salinities.
In this study, plasma osmolality differed across species and locations and, in general, increased with increasing salinity (Fig. 4). However, survival rates at the salinity at which plasma osmolality became isotonic with the osmolality of the salt water (e.g. the highest salinity where plasma osmolality crossed from above to below the diagonal dashed red line in Fig. 4B) did not conform to our expectations. Coastal H. cinerea switched from hypertonic to hypotonic at the highest salinity (9.4 ppt), and in that salinity, ∼72% of coastal H. cinerea larvae were predicted to survive based on model-predicted trend lines. However, inland H. cinerea and inland H. squirella reached their switch point at a lower salinity (8.5 ppt) but had lower 'and' higher survival than coastal H. cinerea, respectively (inland H. cinerea had 59% predicted survival and inland H. squirella had 84% predicted survival). Furthermore, coastal H. squirella and inland H. chrysoscelis switched tonicity at the lowest salinity, 6.5 ppt, but both showed high survival at these salinities (74% survival for coastal H. squirella, and 96% survival for inland H. chrysoscelis). If these data conformed to our previous expectations, we should have observed the highest survival in coastal H. cinerea (which had the highest osmolality switch point) and the lowest survival in inland H. chrysoscelis (which had the lowest osmolality switch point), but this was not the case (Fig. 3). We encourage further inquiry into the relationship between plasma osmolality and saltwater tolerance among amphibians.
We expected divergence in egg deposition patterns among coastal H. cinerea relative to the other species and locations, but we instead observed a high degree of divergence in inland H. squirella populations in oviposition site choice, whereas coastal H. cinerea oviposition patterns conformed to the saltwater avoidance patterns shown by other species. Coastal H. squirella avoided ovipositing eggs into salt water in high salinities, which was mirrored by coastal and inland H. cinerea and H. chrysoscelis. However, H. squirella from inland, salt-naive populations oviposited a greater proportion of their eggs in salt water as salinity increased ( Fig. 1)  when viewed in concert with the data showing reduced hatching probabilities at higher salinities (Fig. 2), is a maladaptive response to saltwater exposure. Further investigation into this puzzling result showed that at least one pair from each inland H. squirella population deposited <75% of their clutch into fresh water in the high-salinity treatments (Table 1), indicating that the pattern was not skewed by a single sampling event or population (Fig. 1). One explanation for the oviposition choices shown by inland H. squirella is that inland populations may lack experience with saline water or may be less capable of detecting differences in salinity. However, the other two species from inland locations also lacked experience with salt water but avoided placing their eggs in salt water. This remains an area for future research.
A detailed understanding of the effects of saltwater exposure on coastal freshwater amphibian communities is required for the development of effective conservation and management strategies. Although we expected salt water to be the primary abiotic filter driving differences in species distributions along a coastal salinity gradient, the factors dictating anuran species ranges along a coastal salinity gradient involve stage-specific, species-specific and location-specific processes that are further mediated by ecological processes and adaptive life history strategies. Therefore, given projected increases in the salinization of freshwater habitats around the globe, the research presented here highlights the need for experimentation to go beyond tolerance assays and integrate information on amphibian habitat use, ecology and physiology across a salinity gradient to best forecast the effects of saltwater intrusion on the structure and function of coastal freshwater communities and identify how salinization affects the geographic distribution of anuran species.